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- PDB-6ep8: InhA Y158F mutant in complex with NADH from Mycobacterium tuberculosis -

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Basic information

Entry
Database: PDB / ID: 6ep8
TitleInhA Y158F mutant in complex with NADH from Mycobacterium tuberculosis
ComponentsEnoyl-[acyl-carrier-protein] reductase [NADH]
KeywordsOXIDOREDUCTASE / mycolic acid biosynthetic process / therapeutic target / isoniazid / mutation / catalytic mechanism / enoyl thioester reductases / tuberculosis
Function / homology
Function and homology information


trans-2-enoyl-CoA reductase (NADH) activity / mycolic acid biosynthetic process / fatty acid elongation / enoyl-[acyl-carrier-protein] reductase (NADH) / enoyl-[acyl-carrier-protein] reductase (NADH) activity / NAD+ binding / peptidoglycan-based cell wall / fatty acid binding / response to antibiotic / plasma membrane
Similarity search - Function
: / Enoyl-[acyl-carrier-protein] reductase (NADH) / Enoyl-(Acyl carrier protein) reductase / NAD(P)-binding Rossmann-like Domain / NAD(P)-binding domain superfamily / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Enoyl-[acyl-carrier-protein] reductase [NADH]
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.8 Å
AuthorsWagner, T. / Voegeli, B. / Rosenthal, R.G. / Stoffel, G. / Shima, S. / Kiefer, P. / Cortina, N. / Erb, T.J.
CitationJournal: J. Biol. Chem. / Year: 2018
Title: InhA, the enoyl-thioester reductase fromMycobacterium tuberculosisforms a covalent adduct during catalysis.
Authors: Vogeli, B. / Rosenthal, R.G. / Stoffel, G.M.M. / Wagner, T. / Kiefer, P. / Cortina, N.S. / Shima, S. / Erb, T.J.
History
DepositionOct 11, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 19, 2018Provider: repository / Type: Initial release
Revision 1.1Sep 26, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 14, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title
Revision 1.3Jan 17, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr2_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Enoyl-[acyl-carrier-protein] reductase [NADH]
hetero molecules


Theoretical massNumber of molelcules
Total (without water)30,24713
Polymers28,8211
Non-polymers1,42612
Water4,756264
1
A: Enoyl-[acyl-carrier-protein] reductase [NADH]
hetero molecules

A: Enoyl-[acyl-carrier-protein] reductase [NADH]
hetero molecules

A: Enoyl-[acyl-carrier-protein] reductase [NADH]
hetero molecules

A: Enoyl-[acyl-carrier-protein] reductase [NADH]
hetero molecules


Theoretical massNumber of molelcules
Total (without water)120,98952
Polymers115,2844
Non-polymers5,70448
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_545-x,-y-1,z1
crystal symmetry operation9_555-x,-x+y,-z+1/31
crystal symmetry operation12_545x,x-y-1,-z+1/31
Buried area28270 Å2
ΔGint-327 kcal/mol
Surface area33150 Å2
MethodPISA
Unit cell
Length a, b, c (Å)98.273, 98.273, 139.836
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number180
Space group name H-MP6222

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Enoyl-[acyl-carrier-protein] reductase [NADH] / Enoyl-ACP reductase / FAS-II enoyl-ACP reductase / NADH-dependent 2-trans-enoyl-ACP reductase


Mass: 28821.057 Da / Num. of mol.: 1 / Mutation: Y158F
Source method: isolated from a genetically manipulated source
Details: The 6-His tag has been cleaved by the thrombin.
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Tissue: / / Cell: / / Gene: inhA, Rv1484, MTCY277.05 / Organ: / / Plasmid: pET15b / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): AI
References: UniProt: P9WGR1, enoyl-[acyl-carrier-protein] reductase (NADH)

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Non-polymers , 5 types, 276 molecules

#2: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Na
#3: Chemical ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE


Mass: 663.425 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Comment: NAD*YM
#4: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-MPD / (4S)-2-METHYL-2,4-PENTANEDIOL


Mass: 118.174 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O2 / Comment: precipitant*YM
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 264 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.3 Å3/Da / Density % sol: 62.8 % / Description: Bi-pyramidal shaped
Crystal growTemperature: 287 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: Thrombin cleaved InhA at 10 mg/ml with 3 to 10 mM of NADH was crystallized in a 24-well crystal plate, Combiclover Junior (Jena Bioscience). The best crystals appeared in drops of 2 ul of ...Details: Thrombin cleaved InhA at 10 mg/ml with 3 to 10 mM of NADH was crystallized in a 24-well crystal plate, Combiclover Junior (Jena Bioscience). The best crystals appeared in drops of 2 ul of enzyme solution, 1 ul of the reservoir solution and 1 ul of distilled water. The reservoir solution contained 100 mM HEPES/NaOH pH 7.5, 50 mM sodium citrate pH 6.5, 7-9 % 2-methyl-2,4-pentanediol. Crystals appeared typically within two to three weeks in this condition.
PH range: 7.5 / Temp details: 287 to 293 K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: BM30A / Wavelength: 1.07244 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Feb 14, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.07244 Å / Relative weight: 1
ReflectionResolution: 1.8→46.61 Å / Num. obs: 37644 / % possible obs: 100 % / Redundancy: 20.5 % / CC1/2: 1 / Rmerge(I) obs: 0.065 / Rpim(I) all: 0.015 / Rrim(I) all: 0.067 / Net I/σ(I): 35.6
Reflection shellResolution: 1.8→1.9 Å / Redundancy: 20.8 % / Rmerge(I) obs: 1.21 / Mean I/σ(I) obs: 2.9 / Num. unique obs: 5382 / CC1/2: 0.924 / Rpim(I) all: 0.271 / Rrim(I) all: 1.24 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(1.10.1_2155: ???)refinement
XDSdata reduction
SCALA3.3.22data scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4TRO

4tro


Resolution: 1.8→42.553 Å / SU ML: 0.18 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 17.87
RfactorNum. reflection% reflection
Rfree0.1751 1884 5.02 %
Rwork0.1454 --
obs0.1468 37562 99.9 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 1.8→42.553 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1995 0 92 264 2351
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0092220
X-RAY DIFFRACTIONf_angle_d1.1293026
X-RAY DIFFRACTIONf_dihedral_angle_d13.1661335
X-RAY DIFFRACTIONf_chiral_restr0.061337
X-RAY DIFFRACTIONf_plane_restr0.007391
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8-1.84870.31111340.23612684X-RAY DIFFRACTION100
1.8487-1.90310.26411420.20442682X-RAY DIFFRACTION100
1.9031-1.96450.22011570.18612687X-RAY DIFFRACTION100
1.9645-2.03470.1991420.16332690X-RAY DIFFRACTION100
2.0347-2.11620.21061350.1512699X-RAY DIFFRACTION100
2.1162-2.21250.15651470.13912706X-RAY DIFFRACTION100
2.2125-2.32910.1671330.12452731X-RAY DIFFRACTION100
2.3291-2.4750.1811420.12422738X-RAY DIFFRACTION100
2.475-2.66610.14211440.1382735X-RAY DIFFRACTION100
2.6661-2.93440.17751320.13282756X-RAY DIFFRACTION100
2.9344-3.35880.15631530.14082780X-RAY DIFFRACTION100
3.3588-4.23110.1581490.13292818X-RAY DIFFRACTION100
4.2311-42.56530.17911740.1552972X-RAY DIFFRACTION99
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.4729-0.01060.06021.97880.95191.09710.08320.1085-0.0802-0.1473-0.21960.3708-0.1211-0.77140.02370.1635-0.0081-0.02030.5943-0.04460.3264-8.3725-40.840216.6173
21.38080.2695-0.06121.5405-0.30431.20940.0084-0.15230.18650.1365-0.03280.3749-0.2292-0.79760.05760.20210.0886-0.00530.6122-0.04230.3469-7.2972-32.508324.881
36.85980.8459-0.28482.5502-1.11611.3444-0.02091.15990.0331-1.16280.22050.77360.058-0.8199-0.00130.62560.0246-0.04780.3960.04380.344216.122-20.89978.5998
41.66120.4317-0.22731.3461-0.1551.69520.0069-0.0350.03810.0325-0.01410.1334-0.1711-0.29410.00140.15020.0208-0.01390.2402-0.02630.22059.067-33.140223.9338
51.06390.01010.47610.93110.57262.0102-0.03950.2360.1968-0.23460.07320.1046-0.2335-0.26060.10420.2282-0.0159-0.02910.36220.03130.32356.8629-37.137911.257
60.738-0.17040.46082.782-0.8552.6216-0.23880.28470.1379-0.34590.0505-0.1565-0.12750.05090.08030.2902-0.0762-0.04330.42260.02010.291713.9298-41.5922.9461
71.14870.01130.45511.26580.11352.1914-0.0010.082-0.0019-0.0242-0.020.04710.1021-0.23980.0140.1688-0.0582-0.00720.2443-0.01060.246310.0385-48.523719.7959
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 2 through 53 )
2X-RAY DIFFRACTION2chain 'A' and (resid 54 through 99 )
3X-RAY DIFFRACTION3chain 'A' and (resid 100 through 112 )
4X-RAY DIFFRACTION4chain 'A' and (resid 113 through 182 )
5X-RAY DIFFRACTION5chain 'A' and (resid 183 through 208 )
6X-RAY DIFFRACTION6chain 'A' and (resid 209 through 235 )
7X-RAY DIFFRACTION7chain 'A' and (resid 236 through 269 )

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